We first sought to determine whether the hippocampal load of Aβ was altered in LTED females subjected to GCI. DAB staining was used to visualize endogenous neuronal Aβ in the STED and LTED hippocampal CA1 region of non-ischemic sham and ischemic (GCI) Pla- and E2-treated animals. The results revealed a robust increase in number of pyramidal cells immunopositive for intracellular Aβ oligomers 24 h post GCI in the hippocampal CA1 region of LTED, but not STED, females (Fig. 1A: e, f and B). Furthermore, Western blotting

analysis also revealed significantly increased Aβ oligomer formation in the hippocampal CA1 region of LTED female rats 24 h post GCI, relative to α-tubulin expression (Fig. 1B), Bortezomib ic50 and this increase was not attenuated by delayed E2 treatment in LTED females. These findings suggest that neuronal Aβ load is increased in long-term surgically menopausal rats subjected

to cerebral ischemia and that delayed E2 therapy cannot prevent this event. Since neurofibrillary tangles are another LDN-193189 purchase major neuropathological hallmark of AD, we next chose to examine E2′s ability to regulate the hyperphosphorylation of tau following chronic loss of ovarian E2. Cerebral ischemia is a well-known tauopathy.35, 36, 37 and 38 In fact, we previously demonstrated that GCI induces significant hyperphosphorylation of tau 24 h post GCI and that low-dose E2 pretreatment attenuates this event.16 We, thus, hypothesized that E2′s regulation of tau hyperphosphorylation may be lost following LTED. To investigate, we examined paired helical filaments (PHF) of microtubule-associated tau phosphorylated at Ser 396 and Ser 404, two residues implicated in human AD neuropathology. Results revealed that both the number of PHF-immunopositive cells (Fig. 2A) and PHF protein levels (Fig. 2B) were increased 24 h after GCI (Fig. 2A: b, e and B), and 1 week of E2 pre-treatment, initiated immediately following ovariectomy, was

able to prevent this event in STED rats (Fig. 2A: c and B). In contrast, delayed E2 treatment was unable to mitigate the phosphorylation of tau at these two pathological residues in LTED rats (Fig. 2A: f and B), suggesting that E2 regulation of tau phosphorylation substrate level phosphorylation is, indeed, lost following LTED. To better understand the mechanisms underlying the marked elevation of endogenous Aβ in LTED rats, we next examined hippocampal CA1 expression of two putative α-secretases: ADAM 10 and ADAM 17, as well as the β-secretase BACE1 (section 3.3). ADAM 10 and ADAM 17 are thought to be the driving forces of non-amyloidogenic processing of APP, and although some controversy exists regarding which putative α-secretase is mainly responsible,9 recent studies have provided evidence that ADAM 10 is the primary α-secretase and that ADAM 17 plays a more secondary role in the non-amyloidogenic processing of APP.